Assay method for detecting presence of bacteria

ABSTRACT

An assay method is provided to easily and quickly detect the presence of organisms capable of being cultured, such as bacteria, characterized by antibody capture of the organism of interest by use of specialized magnetic beads, incubation of the captured cells to form colonies; removal of material from the colonies with a colony lift membrane; and detection of the colony material on the membrane sheet by use of labeled antibodies, PCR or nucleic acid probes.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 07/999,363,filed on Dec. 31, 1992, now U.S. Pat. No. 5,491,068 which is acontinuation-in-part of application Ser. No. 07/654,967, filed on Feb.14, 1991, (now abandoned) the entire contents of which are herebyincorporated by reference.

FIELD OF THE INVENTION

This invention relates to an assay method for quickly and easilydetecting the presence of bacteria or other culturable organisms. Inparticular, an immunoassay method is utilized to detect the presence ofviable bacteria in foods and other potentially contaminated samplesusing an assay characterized by 1) capture of specific bacterial cellswith specific antibodies immobilized on magnetic beads; (2) incubationof the captured cells by the beads to form bacterial colonies; (3)imprint of the colonies to a colony lift membrane; and (4) confirmationand quantification of the colonies on the colony lift membrane.

BACKGROUND OF THE INVENTION

The presence of bacterial pathogens is a well recognized cause of severeillness, so that there is an ever present need for the detection of suchpathogens in both clinical specimens (i.e. blood, tissue, urine andother body extracts and fluids), agricultural specimens (such as foodproducts) and environmental specimens (such as surfaces in foodprocessing plants).

However, current tests for the detection of bacterial pathogens, such asin food, typically require a number of days to complete. During thisperiod of time, between sampling and assay determination, fresh food anddairy products will enter the food chain and therefore be consumed bythe public. If a test indicates the presence of pathogens, expensiveproduct recalls may result, or, worse, before the test results arediscovered an outbreak of sickness may occur.

As stated above, traditional methods to detect the presence of bacterialfood pathogens require an extended period of time, basically due to theneed for an enrichment/incubation period. This incubation/enrichmentperiod is intended to allow for growth of these bacteria from abackground of competing microorganisms and an increase in bacterial cellnumbers to more readily aid in identification. In many cases a series oftwo or three separate incubations is needed to isolate the targetbacteria. However, such enrichment steps can actually compromise testsensitivity by killing some of the cells sought to be measured.

In the standard FDA procedure for detection of Listeria in food products(Bacteriological Analytical Manual, 7th ed., 1992; Chapter 10 25 g or 25ml of a food sample is mixed with 225 ml of enrichment broth. Thissample in broth mixture is incubated for 2 days. At the end of days 1and 2 a sample of the broth culture is streaked onto petri platescontaining selective growth agar and these plates are incubated for anadditional 1-2 days. Identification of Listeria colonies is done by eye.This identification, however, is subjective, and presumed colonies mustbe confirmed by additional tests, which require another 1-2 days.Because of growth of bacteria during the enrichment step, the number ofcolonies on the agar plates does not represent the number of bacteria inthe original sample. This test can only detect the presence of bacteria,and cannot quantitate the numbers originally present in the sample.

More recent methods of bacteria detection in food products haveattempted to reduce the time needed for enrichment or confirmation. Manyof these procedures utilize antibodies. A typical procedure, exemplifiedby the Listeria-Tek and Salmonella-Tek assays (Organon Technica Corp.),is a two site assay. That is, one antibody is immobilized in amicrotiter well and acts to capture the target bacteria. This allows forseparation of the target bacteria from the sample. A second antibodylabelled with an enzyme is used to detect the captured bacteria.Theoretically, such an assay could be used to detect bacteria directlyin a food sample. The actual sensitivity limit of these assays, however,makes it necessary to culture the target bacteria from the food sample.Because of the need for enrichment, the assay still requires 24-48hours, even though the confirmation step is reduced to 1-2 hours.Enrichment also makes quantitation impossible.

Another alternative method for Listeria detection is immunomagneticisolation. In this procedure, antibodies to the bacteria of interest areimmobilized on magnetic beads. The beads, with attached antibodies,interact with the target organisms, which can be separated from othersample material and microorganisms in a magnetic field. This procedureis intended to reduce or eliminate the 24-48 hours enrichment period.Production and use of magnetic beads have been described in U.S. Pat.Nos. 3,970,518 (Giaever), 4,230,685 (Senyi and Widder), 4,677,055 (Dodinet al.), and 4,695,393 (Whitehead et al.). Immunomagnetic beads havebeen used to isolate Salmonella (Vermunt et al., J. Appl. Bact. 72, 112,1992), Staphylococcus aureus (Johne et al., J. Clin. Microbiol. 27,1631, 1989) and Listeria (Skjerve et al., Appl. Env. Microbiol. 56,3478, 1990) from foods, and Escherichia coli from fecal samples (Lund etal., J. Clin. Microbiol. 29, 2259, 1991). In all of these examples,immunomagnetic capture is very inefficient at low numbers of bacteria.At the low levels of bacteria significant in food microbiology (<100bacteria per gram), these methods cannot be used without enrichment.Interference by non-target organisms sometimes occurs, requiring theaddition of selective enrichment, or a confirmation step, or both, tomake a complete assay.

In summary, these existing "rapid" immunoassay procedures for bacteriadetection in food samples all require at least one dilution of sampleinto growth medium, followed by an enrichment period, then an assayprocedure which only utilizes a fraction of this final culture. Theactual assay sample thus only corresponds to a small fraction of theoriginal food sample. The bacterial culture step, or steps, musttherefore overcome this dilution factor, adding to the amount of neededculture time. The use of the enrichment step also makes quantificationof bacteria impossible. In addition, the utilized enrichment steps maykill the bacteria sought to be identified, producing a high falsenegative rate.

The method of the present invention overcomes the low capture efficiencycharacteristic of all previous immunomagnetic procedures. Immunomagneticcapture of bacteria is a complex process, and several parameters areimportant in successful capture. Good recovery of bacteria from foodsamples requires strong interaction between the bacteria and theimmunomagnetic beads. The higher efficiency of the assay of the presentinvention results from improvements in both particle design and themethod used for magnetic isolation.

A combination of bead porosity, bead size, attachment method, and longcapture time accounts for the improved performance. The improvement incapture efficiency is such that bacteria can be isolated directly fromfood samples, without enrichment, at levels of less than 10 bacteria pergram of food. Because enrichment is avoided, the method allowsquantitation of the number of bacteria present. In addition, thebacterial colonies formed are individually confirmed as target bacteriaby an immunochemical confirmation step.

OBJECTS AND SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide a methodfor the detection of viable bacteria which eliminates the selectiveculturing steps.

It is another object of the invention to provide a method for quicklyconfirming and quantitating the presence of any culturable organism,particularly bacteria, including potentially pathogenic bacteria.

It is a further object of the invention to provide a method for thedetection of bacteria by which the detection can be easily made byviewing with the human eye.

These and other objects of the invention are accomplished by providing amethod wherein:

(1) the bacteria cells of interest are selectively captured and removedfrom a sample by the use of an antibody bound to magnetic beads;

(2) the captured bacteria cells are spread on a medium to form colonies;

(3) the bacteria colonies are contacted with a colony lift membrane toattach colony material to the membrane; and

(4) the presence of colony material from the colonies of the bacteria ofinterest is detected by use of one of various procedures, including DNAor RNA probes, PCR techniques and labeled antibodies which provideevidence of the presence of the bacteria of interest.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, one object of the invention is to provide an assayprocedure for rapid and easy identification of the presence of aparticular bacteria of interest in a sample. The assay procedure isbroadly applicable to the detection of any culturable organism,including bacteria, molds and yeast. Of particular importance is thedetection of the presence of potentially harmful contaminants,particularly those which cannot be visually detected by eye. A broadrange of contaminants can be detected by the assay, so long as thecontaminant can be cultured to form colonies and antibodies can beraised against the contaminant, DNA or RNA probes can be used to detectthe contaminant or PCR techniques can be used to detect DNA materialfrom the contaminant.

In a particular preferred aspect, the assay is used to detect variousbacteria, and can be utilized to detect the presence of any specific,selected bacteria of interest. The bacteria can be either pathogenic ornon-pathogenic, although the invention is particularly important fordetection of potentially contaminating pathogenic bacteria. Specificbacteria detectable by the assay of the invention include, for example,Listeria, Campylobacter, Escherichia coli, Salmonella, Clostridia (suchas Clostridium botulinum and Clostridium perfingens), Shigella,Staphylococci (such as Stapylococcus aureus), Vibrio (such as Vibriovulnificus, Vibrio cholerae and Vibrio parahaemolyticus), Yersinia (suchas Yersinia enterocolitica and Yersinia pseudotuberculosis) Plesiomonasstrigelloides, Bacilli (such as Bacillus cereus) and Aeromonas (such asAeromonas hydrophila).

In addition, various molds can be detected, including Byssochlamys,Fusarium, Geotrichum, Penicillium and Scopulariopsis and various yeastscan be detected, such as Kluyveromyces, Pichia, Saccharomyces, Candidaand Rhodotorula.

The method of the invention, therefore, generally comprises thefollowing procedural steps:

1) The sample to be tested is, if necessary, liquified or otherwiseprepared for use in the assay.

2) The liquid sample is combined with magnetic beads having immobilizedthereon monoclonal or polyclonal antibodies to the selected bacteria ofinterest, the presence of which is to be determined by the assay. Ifpresent, the target selected bacteria cells are thereby immobilized ontothe immunomagnetic beads. The immunomagnetic beads with attachedimmobilized bacteria are then washed to remove any remaining sample.

3) The immunomagnetic beads with the immobilized bacteria are thenspread on a culture medium, and the bacteria are allowed to grow to formcolonies.

4) To confirm that the colonies are the bacteria of interest, thecolonies are then contacted with a colony lift membrane, and themembrane is removed, whereby the colony material from the bacterialcolonies is attached to the lift membrane.

5) The colony material attached to the lift membrane is then subjectedto one of several detection/analytical procedures by which the presenceof the bacteria of interest can be either qualitatively orquantitatively determined. These detection steps include: (a) use ofnucleic acid probes; (b) use of PCR; and (c) use of antibodies.

(a) If nucleic acid probes are used, the lift membrane (nitrocellulose)is placed in a lysis buffer to allow lysis of the bacterial colonies andrelease bacterial nucleic acids which become bound to the lift membrane.The membrane is then blocked and hybridization is carried out directlyin the blocking solution by adding to it a RNA or DNA probe specific forthe bacteria of interest. The probe is, for example, labeled withfluorescein, so that, the hybridized membrane can then be incubated withan anti-fluorescein antibody labelled for subsequent detection.

(b) If PCR detection is to be used, again the lift membrane havingcolony material attached thereto is added to a lysis buffer to releasenucleic acids. The released nucleic acids are then precipitated,collected and form the template for a PCR reaction. The template nucleicacid is appropriately mixed with nucleoside triphosphates, divalentcations and primers.

(c) If antibody detection is to be used, the lift membrane is treatedwith a fixing agent to fix the colony material to the membrane and themembrane is blocked to lower non-specific reactivity. The membrane,having fixed thereto material from the bacterial colonies, is thentreated with a labeled or unlabeled first antibody specific to thebacteria of interest to be detected, and the membrane is washed toremove any unbound first antibody. If the first antibody was unlabeled,the membrane is then treated with a second antibody which is labeled andis specific for the first antibody, and the membrane is washed to removeany unbound labeled second antibody. The presence of labeled first orsecond antibody on the membrane is then detected, specifically by meansto permit visual evidence of the presence of colonies of the bacteria ofinterest.

As can be seen from the above method scheme, the method of the inventionis particularly characterized by the use of immunomagnetic beads tofirst select out bacteria from a sample. The beads must be capable ofefficiently capturing bacteria from foods at realistic levels, while notcapturing other bacteria which may be present at much greater numbers.The antibody used for this step need not be totally specific to thebacteria of interest as subsequent selective and specific steps combineto confer the ultimate specificity of the assay for only specificbacteria of interest. Further uniquely characterizing the method of theinvention is the use of a colony lift membrane and subsequentconfirmation steps. Using these characteristic features, the method ofthe invention can be importantly applied to the rapid and specificdetection of viable strains of bacteria, particularly detection by asimple visual means, detectable by the human eye. The assay particularlyprovides a very sensitive assay detection, capable of detecting 1 colonyforming unit (CFU) per 25 ml of sample, more than 100 times moresensitive than prior immunomagnetic methods.

High sensitivity and efficiency of the assay of the invention areaccomplished by an important combination of factors relating to thedesign and use of magnetic beads having antibodies attached thereto.

First, the antibodies must be accessible at the surface of the magneticbeads, so nonporous beads are required. Giaever, Senyi, and Dodin (citedabove) all disclose the use of porous magnetic beads. Second, theantibodies must be oriented with their binding sites outward. In oneembodiment, attachment of antibodies to magnetic particles isaccomplished through a protein A intermediate. That is, protein A isfirst attached to the magnetic particles and the antibodies of choiceare then bound to the protein A. The use of the protein A intermediategreatly increases the effectiveness of capture by the attachedantibodies (Forsgren et al., J. Immunol. 99, 19, 1976). Protein Aattaches to the Fc portion of IgG subclass antibodies, thus extendingand presenting the Fab portion of these antibodies. The resultingcorrect orientation of the antibodies and extension away from theparticles leads to a very effective interaction between the boundantibodies and their target. Senyi describes protein A magnetic beads,but without the other improvements described here, those would not besuccessful. Third, the magnetic particles must be in the submicron sizerange, which creates a large surface area. Skjerve used solidpolystyrene beads of 2.8 μm diameter, and these were much lessefficient. Fourth, contact time between the beads and the bacteria mustbe long enough to allow strong interaction. Magnetic beads are mixedwith sample for 30 minutes to 2 hours, a longer time than reported inprevious trials of magnetic beads.

Step 1: Liquefaction

The assay of the invention can be utilized to detect the presence ofbacteria in a wide variety of samples, both solid and liquid, includingfood, agricultural products, environmental samples and various clinicalspecimens. If the sample is liquid, it can be per se subjected to theprocedure of the invention, or first diluted, or concentrated bycentrifugation. On the other hand, if the sample is solid, it should befirst liquified (for example in water) using standard known techniques,such as by use of a blender or stomacher. The liquid or liquified samplemay, if desired, be filtered through a course paper, glass or othermatrix filter to remove particulates. If the sample to be tested is anenvironmental sample, then swabs or scrapings of the tested surface ormaterial are mixed in a collection buffer and then treated as aliquified food sample.

Step 2: Immobilization With Antibodies

Antibodies (polyclonal or monoclonal) to target bacterial cells areimmobilized on magnetic beads and used to separate the bacteria cellsfrom the sample. In this step, one or more antibodies may be used torecognize all target strains of the bacteria genus of interest. Theantibodies used in this step recognize bacteria cell surface antigens.After an incubation period in which the liquid sample or liquifiedsample is mixed with the magnetic particles having antibodies attached,the antibody bound particles and attached bacteria are then separatedfrom the sample by means of a magnetic field (magnetic capture) andwashed to remove other potential impurities.

As the antibodies for capture, and, as discussed later, for detection,of the bacteria, any class of antibodies can be used (including IgG andIgM) and either polyclonal antibodies or monoclonal antibodies can beused depending upon various factors, including the degree of sensitivitydesired. If polyclonal antibodies are to be used, then such antibodiescan be prepared according to per se known procedures. For example,procedures such as those described by Hurn, B. A. et al. (1980) in Meth.in Enzymology, Ed. Van Vanakis, H. and Langone, J., pp. 104-142, can beused. The preparation of monoclonal antibodies is known and ifmonoclonal antibodies are to be used in this invention, they areprepared using the method originally authored by Milstein and Kohler andpublished in Nature (1975), 256, pps. 495-497.

The magnetic beads must meet several requirements in order to be usefulin this assay. They must be nonporous, so that the antibody moleculesremain on the surface of the bead, where they an contact the bacteria.The magnetic beads described in U.S. Pat. Nos. 3,970,518 (Giaever),4,230,685 (Senyi and Widder), and 4,677,055 (Dodin et al.) are allporous, allowing antibody molecules to enter the beads. Since thebacteria cannot enter the bead, the amount of antibody available tocontact bacteria is reduced. In addition, the beads must be less thanabout 1 μm in diameter, so that the beads are similar in size as, orsmaller than, the bacteria. The magnetic beads used by Skjerve (citedabove), commercially available from Dynal, were 2.8 μm. Their lowcapture efficiency was due in part to the relatively large beaddiameter.

Beads for use in the present invention, therefore, are non-porous andhave a diameter of from about 50 nm to about 1 μm, preferably from 0.3to 1 μm. The beads must also contain a chemically functional groupthrough which the antibody can be attached. The beads described byWhitehead et al. (U.S. Pat. No. 4,695,393) are suitable. Whitehead,however, does not describe capture of bacteria, and gives no indicationthat their beads would be an improvement over the larger or porous beadsdescribed earlier. Other suitable beads include protein coated magnetitebeads, such as beads coated with bovine serum albumin which may beadditionally coated with an antibody-linking compound like Protein A.Useful beads are described, for example in PCT Patent Publication WO9102811 to Immunicon Corp. and are commercially available as ImmuniconProtein A Magnetic Separation Media, Cat. No. G6100.

Attachment of the antibody to the magnetic beads must be such that thebinding portion of the antibody extends away from the surface of thebead, allowing contact between the binding portion of the antibody andthe bacteria. To do this, the antibody is attached at some site on itsnonbiding portion. Potential sites for direct covalent biding includethe carbohydrate portion and sulfhydryl groups between the heavy chains.The antibody can also be attached indirectly using a ligand or proteinwith affinity for the nonbinding portion, such as another antibody orprotein A. Binding via protein A is a preferred method of thisinvention. The method of attachment of protein A to magnetic particlesmay proceed by any of several processes available through the scientificliterature. In one such procedure, magnetic iron oxide particles ofapproximately 1 μm diameter, derivatized with an amino group, arechemically activated with glutaraldehyde. The activated magneticparticles are then mixed with protein A resulting in a magnetic particleto which protein A is covalently attached. The antibodies are then addedto the protein A magnetic particles and after a short incubation theprotein A-antibody complexes form (Weetall, Meth. Enzymol. 44, 134,1976). These derivatized particles with protein A-antibodies attachedare now ready for use in bacterial cell capture.

During the capture step, strong interactions must form between theantibodies and the bacteria. A short incubation time, such as 5 minutes,allows only weak interactions to form, producing low capture efficiency.At least 30 minutes, and preferably 2 hours, is needed to allowsufficiently strong interactions to produce high capture efficiency.

Step 3: Growth of Bacterial Colonies

The captured and immobilized bacterial cells are spread on a medium onwhich the cells will grow and are incubated. Incubation is conducted fora time sufficient to form bacterial colonies visible to the eye.Spreading the cells produces separate colonies from the individualcells, providing a means of quantification.

Particular media for incubation depend, of course, upon the bacteria ofinterest to be detected. Such mediums, preferably solid, are per seknown to those skilled in the art for various bacteria, as disclosed,for example, in the Bacteriological Analytical Manual. Incubation timesand conditions are also varied and per se known, depending upon theparticular bacteria of interest. Generally, sufficient growth isaccomplished within 6 to 24 hours.

Alternatively, the separated bacteria can be grown in a liquid culturemedium. This removes the possibility of quantitation, but still allowssubsequent confirmation of the presence of the selected bacteria.Confirmation in this case can be by traditional biochemical tests, or byimmunochemical or nucleic acid probe techniques.

Step 4: Colony Lift onto Membranes

After incubation of the bacteria is completed, confirmation of thespecific bacteria of interest is still necessary. This can be done invarious ways. For example, colonies can be confirmed by traditionalbiochemical methods or by one of the newer immunochemical or nucleicacid probe tests. The preferred form of this invention usesimmunochemical confirmation on a colony lift membrane, in order topreserve the quantitative capabilities provided by the efficientmagnetic capture step.

A colony lift membrane is placed in contact with the growth medium,whereby the colonies attach and imprint colony material to the membrane.Colony lift membranes are per se known and may be comprised of, forexample, nitrocellulose or nylon. The membrane is preferably cut to thesize of the container or dish containing the growth medium, so that allcolonies growing on the container are overlayed with the same singlesheet. In this manner, the sheet/membrane acquires the same pattern ofcolonies that was originally contained on the growth medium.

Step 5: Detection of Bacteria of Interest

The lift membrane having colony material can now be subjected to one ofseveral procedures for detection of the bacteria of interest.

a) Detection by use of nucleic acid probes:

Detection of the bacteria of interest can be performed by use of nucleicacid probes following procedures which are per se known. Suitableprocedures for such detection of Listeria are described, for example, inU.S. Pat. No. 5,089,386, PCT Patent Publ. WO 90/08841, PCT Patent Pub.WO 92/15883, and PCT Patent Pub. WO 89/06699, each of which is herebyincorporated by reference.

A suitable nucleic acid probe assay is comprised generally of (1) sampletreatment and lysis; (2) hybridization with the selected probe(s); (3)hybrid capture and (4) detection.

Lysis of the bacteria is necessary to release the target molecules forthe probes. The nucleic acid target molecules are released by treatmentwith any of a number of lysis agents, including alkali (such as NaOH),guanidine salts (such as guanidine thiocyanate), enzymes (such aslysozyme, mutanolysin and proteinase K), and detergents.

Lysis of the bacteria, therefore, releases both DNA and RNA,particularly ribosomal RNA and chromosomal DNA both of which can beutilized as the target molecules with appropriate selection of asuitable probe. Use of rRNA as the target molecule(s), however, ispreferred because rRNAs constitute a significant component of cellularmass, thereby providing an abundance of target molecules. The use ofrRNA probes also enhances specificity for the bacteria of interest, thatis, positive detection without undesirable cross-reactivity which canlead to false positives or false detection.

Hybridization is comprised of the addition of the specific nucleic acidprobes. In general, hybridization is the procedure by which twopartially or completely complementary nucleic acids are combined, underdefined reaction conditions, in an anti-parallel fashion to formspecific and stable hydrogen bonds. The selection or stringency of thehybridization/reaction conditions is defined by the length and basecomposition of the probe/target duplex, as well as by the level andgeometry of mispairing between the two nucleic acid strands. Stringencyis also governed by such reaction parameters as temperature, types andconcentrations of denaturing agents present and the type andconcentration of ionic species present in the hybridization solution.

The hybridization phase of the nucleic acid probe assay is performedwith a single selected probe or with a combination of two, three or moreprobes. Probes are selected having sequences which are homologous tounique rRNA sequences of the target organism. Suitable probes useful forthe detection of Listeria are described, for example, in U.S. Pat. No.5,089,386, PCT Patent Pub. WO 90/08841 and PCT Patent Pub. WO 89/06699and are commercially available, for example, from Gene-Trak Systems. Ingeneral, a first capture probe is utilized to "capture" formed hybridmolecules. The hybrid molecule is then detected by use of antibodyreaction or by use of a second detector probe which may be labelled witha radioisotope (such as phosphorus-32) or a fluorescent label (such asfluorescein) or chemiluminescent label.

b) Detection by use of PCR:

Detection of the bacteria of interest can also be performed by use ofPCR techniques. A suitable PCR technique is described, for example, inPCT Patent Pub. WO 92/08805. Such detection procedures are applied tothe colony material adhered to the lift membrane or, alternatively,directly to the bacteria captured on the magnetic beads. In either case,the bacteria is combined with a lysis buffer and collected nucleic acidtarget molecules are then utilized as the template for the PCR reaction.

c) Detection by use of antibodies:

For detection of the selected bacteria by use of antibodies, after thecolony material is adhered to the lift membrane, it is then necessary tofix the colony material to the lift membrane by treatment with anappropriate fixing agent in order to ensure that the colonies adheremore firmly to the sheet. Suitable fixing treatments include placing thelift membrane in a solution of methanol or placing the membrane in asolution of the detergent sodium dodecyl sulfate with brief heating to70° C.

It may also be desirable to treat the fixed colonies and membrane withper se known blocking agents to prevent non-specific reactivity of thesubsequent detecting antibodies. Suitable blocking agents include, forexample, casein and BSA.

The lift membrane having fixed thereto the colony material from thebacterial colonies is next contacted with antibodies specific to thebacteria of interest. As noted above, either polyclonal or monoclonalantibodies can be utilized, but in either case have affinity for theparticular bacteria to be detected. These antibodies, when contactedwith the lift membrane, will adhere/bind to material from the specifictarget bacteria colonies, but will not bind to the other colonies.

Useful polyclonal antibodies include, for example, those from Difo polysera raised in rabbits. These antibodies can also be specific forparticular strains to be detected. For example, Listeria monocytogenesincludes strains of serovars 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4a/b, 4b,4c, 4d, 4e, 7, with strains of serovars 1/2a, 1/2b and 4b being the mostcommon pathogenic strains. It is useful, therefore, to detect thepresence in a sample of pathogenic Listeria by utilizing a polyclonalsera against Listeria serovars 1/2 and 4b. Other useful antibodies,polyclonal or monoclonal, are those specific for proteins orcarbohydrates on the cell surface of the bacteria of interest. Ofparticular usefulness are monoclonal antibodies against Listeriateichoic acids of serovars 1/2 and 4b.

Treatment as described above with a first antibody, specific to thebacteria cells of interest, provides the first step for selection andidentification of the specific bacteria of interest. The membrane sheetis then contacted with a second antibody. Again, this antibody may beeither polyclonal or monoclonal, but importantly is (a) capable ofbinding to the first antibody and (b) labeled in a manner to enablesubsequent detection.

If the first antibody is, for example, the above-noted Difco poly sera,then the second antibody is an anti-rabbit IgG/label conjugate. If thefirst antibody is a monoclonal antibody derived from mouse, than thesecond antibody is an anti-mouse IgG/label conjugate.

Alternatively, the first antibody (or the first capture nucleic acidprobe described above) can itself be labelled. The assay can thenproceed to detection of the label without the need for use of a secondantibody (or a second detector probe).

With respect to labeling of the antibodies, these are labeled eitherdirectly or indirectly with labels used in other known immunoassays.Direct labels may include fluorescent, chemiluminescent, bioluminescent,radioactive, metallic, biotin or enzymatic molecules. Methods ofcombining these labels to antibodies or other macromolecules are wellknown to those in the art. Examples include the methods of Hijmans, W.et al. (1969), Clin. Exp. Immunol. 4, 457-, for fluoresceinisothiocyanate, the method of Goding, J. W. (1976), J. Immunol. Meth.13, 215-, for tetramethylrhodamine isothiocyanate, and the method ofIngrall, E. (1980), Meth. in Enzymol. 70, 419-439 for enzymes.

These detector antibodies may also be labeled indirectly. In this casethe actual detection molecule is attached to a secondary antibody orother molecule with binding affinity for the anti-bacteria cell surfaceantibody. If a secondary antibody is used it is preferably a generalantibody to a class of antibody (IgG and IgM) from the animal speciesused to raise the anti-bacteria cell surface antibodies.

For example, the second antibody may be conjugated to an enzyme, eitheralkaline phosphatase or to peroxidase. To detect the label, after themembrane sheet is contacted with the second antibody and washed, themembrane sheet is immersed in a solution containing a chromogenicsubstrate for either alkaline phosphatase or peroxidase. A chromogenicsubstrate is a compound which can be cleaved by an enzyme to result inthe production of some type of detectable signal which only appears whenthe substrate is cleaved from the base molecule. The chromogenicsubstrate is colorless, until it reacts with the enzyme, at which timean intensely colored product is made. Thus, material from the bacteriacolonies adhered to the membrane sheet will become an intenseblue/purple/black color, or brown/red while material from other colonieswill remain colorless. Examples of detection molecules includefluorescent substances, such as 4-methylumbelliferyl phosphate, andchromogenic substances, such as 4-nitrophenylphosphate,3,3',5,5'-tetramethylbenzidine and 2,2'-azino-di-3-ethelbenz-thiazoliane sulfonate (6)!. In addition to alkalinephosphatase and peroxidase, other useful enzymes includeβ-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase,α-mannosidase, galactose oxidase, glucose oxidase and hexokinase.

EXAMPLE 1 Detection of Listeria Utilizing Antibody Detection

The following is a specific example to show the use of the assay of theinvention to particularly detect the presence of Listeria.

1. To magnetic particles approximately 0.3 μm in size with covalentlyattached protein A (such as Immunicon Protein A beads), add antibodiesto Listeria (such as Lee Labs, Inc. Listeria O antiserum Poly 1,4).

2. Obtain sausage sample known by conventional microbiological methodsto contain 100,000 to 1 million bacteria per gram and which contain 23Listeria per gram, as determined by the FDA Most Probable Number Assay(MPN), and 2.3 Listeria per gram, as extrapolated from the FDA MPNassay. The MPN assay is accurate within one order of magnitude. Theimmunomagnetic capture procedure recovers 30% of Listeria from sausagesamples.

3. Liquify the above samples as follows: add 25 grams sausage to 150 mLphosphate buffered saline (PBS)+0.05% Tween20, and stomach for twominutes at normal speed.

4. Filter the stomachate so that 40 mL filtrate is collected.

5. Centrifuge the 40 mL filtrate at 11,000×g for 15 minutes toconcentrate the microorganisms. Decant the supernatant and resuspend thepellet in 2 mL PBS+Tween.

6. Combine the 2 mL resuspension, equivalent to 5.7 grams of theoriginal sausage sample, with 100 μL of the magnetic beads havingimmobilized thereon antibodies to Listeria.

7. After an incubation time of 2 hours rotating end to end, place themixture in a magnetic field to remove the magnetic beads having Listeriacells immobilized thereon, and wash the beads a total of two times withPBS to remove undesirable impurities and unbound material or cells.

8. Spread the separated magnetic beads on the surface of a petri dishcontaining brain-heart infusion agar, plus lithium chloride andceftazidime.

9. After an incubation of 20 hours at 37° C., examine the plates forgrowth of bacterial colonies. In this example, duplicate plates from the23 Listeria CFU/g per gram sausage sample contained 29 and 26Listeria-like colonies, as well as several non-Listeria-like colonies.Plates from the 2.3 Listeria per gram sausage sample contained 8 and 2Listeria-like colonies.

10. To confirm these suspected colonies as Listeria, place a colony liftmembrane (e.g. Pall Biodyne Transfer Membranes) onto the plate of solidgrowth medium to obtain an imprint of the colonies on the membrane. Wait5 minutes before peeling the membrane from the plate. The media platesmay then be further incubated or stored at 4° C. for use in furthertesting.

11. Place the colony lift membrane, colony side up, into a traycontaining 10 mL methanol for 5 minutes to kill bacteria and to fixantigens onto the membrane surface so that they will not wash off.

12. Wash the membrane under a fast flowing water tap to remove anyexcess colony material.

13. Wash the membrane with PBS+Tween for 2-5 minutes. Remove wash fluidand repeat wash two more times, for a total of three washes.

14. Block the membrane to prevent nonspecific reactivity of thedetecting antibodies by placing the membrane in 10 mL PBS+Tween+2%nonfat dry milk. Shake on a reciprocating shaker for 30 minutes.

15. After the 30 minute incubation, pour off any remaining solution andwash the membrane a total of three times with PBS+Tween.

16. Incubate the membranes with 10 mL of first antibody in PBS+Tweenwhile shaking for 30 minutes. The antibody for this example is a mousemonoclonal IgM capable of binding to Listeria cell surface proteinantigens fixed to the membrane.

17. After the 30 minute incubation, pour off the first antibody solutionand wash the membrane a total of three times with PBS+Tween.

18. Incubate the membrane with 10 mL of second antibody (goat anti-mouseIgM antibody conjugated to alkaline phosphatase) diluted in PBS+Tween,while shaking for 30 minutes. The second antibody is capable of bindingto the first antibody, which is specific to Listeria, so that color isproduced on the membrane at places where antigens from the Listeriacolonies are present.

19. Pour off the second antibody solution and wash the membranes with 10mL water, for a total of three washes.

20. Incubate the membranes with 10 mL of alkaline phosphatate substratesolution on the shaker until purple dots appear on the membrane. Atypical incubation requires about 5 minutes.

21. Stop the reaction by pouring off the substrate and washing with 10mL water per membrane, for a total of two washes.

22. The number of Listeria is then quantitated by counting the number ofpurple signals formed on each membrane. In this example the 23 CFU/gsamples' membranes contained 29 and 26 purple signals, while the 2.3CFU/g samples' membranes contains 8 and 2 purple signals correspondingto the Listeria-like colonies on the media plates, thus confirming thatthey are Listeria. No purple signals were present at the positionscorresponding to the non-Listeria-like colonies.

23. The original contamination level determined by Listertest iscalculated. The sausage samples containing 23 CFU/g, as determined byMPN, contained 16 CFU/g as determined by this method. The sausagesamples containing 2.3 CFU/g, as determined by MPN, contained 2.9 CFU/gas determined by this method.

EXAMPLE 2 Listeria Detection Utilizing Nucleic Acid Probe Detection

Selective capture of Listeria is performed the same as in Example 1 upto the stage at which the colony lift membrane is used to obtain animprint of the colonies (step 10). For this procedure a lift membrane iscomprised of nitrocellulose (NC). The NC membrane is laid over colonieson the master plate for five minutes to make an imprint. The imprintedmembrane is then placed in a lysis buffer (1% (w/v) sodium dodecylsulfate in water) at 37° C. for 30 minutes, to allow lysis of bacterialcolonies and the release of bacterial nucleic acids, which become boundto the NC membrane. This step is followed by a gentle wash in roomtemperature buffer, followed by fixing of the DNA onto the NC membraneby heating the dry membrane at 70° C. for 2 hours. The NC membrane isthen blocked with 0.1% (w/v) bovine serum albumin in wash buffer.Hybridization is carried out directly in the blocking solution by addingto it a DNA probe specific for Listeria species or Listeriamonocytogenes and labeled with fluorescein. Hybridization is carried outat 37° C. for one hour.

The NC membrane is then washed at 37° C. for five minutes, and at 65° C.for five minutes, using the wash buffer. The NC membrane is thenincubated with an antifluorescein antibody labelled with alkalinephosphatase. Alternatively the probe is a biotinylated DNA probe anddetection is by use of avidin-labelled enzyme. The NC membrane isincubated with substrate, bromo-chloro-indoyl-phosphate andnitro-blue-tetrazolium, until blue dots appear over colony material orthe background becomes pale blue. If Listeria are present, there iscolor formation on the membrane corresponding to colonies of Listeria onthe master plate.

EXAMPLE 3 Listeria Detection Utilizing PCR

As noted above, PCR detection can be applied to either the bacteria ascaptured on the magnetic beads or to the bacteria colony materialadhered to the lift membrane. In either case, the bacteria, either onthe beads or from a colony, are mixed with a lysis buffer.

The lysis buffer contains the enzymes lysozyme and mutanolysin. Theseenzymes digest bacterial cell walls so the nucleic acids can bereleased. The sample in lysis buffer is incubated at 37° C. for 30minutes. Then a small amount of lysis buffer containing proteinase K anddetergent is added. Proteinase K digests protein. After a briefincubation, sodium acetate and absolute ethanol are added, toprecipitate nucleic acid. The precipitated nucleic acid is centrifugedto collect it, and dried. This material forms the template in the PCRreaction.

The PCR reaction is begun by mixing template nucleic acid withnucleoside triphosphates and divalent cation, along with primer. Manysequences have been published for Listeria primers in PCR, any of whichis suitable. TAC polymerase, a genetically engineered form of DNApolymerase, is added to the mixture and the solution is covered withmineral oil to prevent evaporation. The mixture is subjected to 30cycles of PCR, followed by electrophoresis. DNA can be visualized byethidium bromide or another type of visualization method. One picogramof template DNA is sufficient to produce detectable levels of amplifiedproduct under these conditions.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included within the scope of the following claims.

We claim:
 1. A method for detecting the presence of an organism capableof being cultured which comprises:a) combining a sample to be tested forthe presence of a selected organism with a magnetic solid support havingimmobilized thereon antibodies to said selected organism, to therebycapture cells of said selected organism from said sample, said magneticsolid support being non-porous magnetic beads having a diameter of from50 nm to 1 μm; b) exposing to a magnetic field said magnetic beadshaving bound thereto said antibodies and captured selected organisms tothereby separate said magnetic solid support from said sample; c)treating said magnetic beads to detect the presence of said selectedorganism.
 2. The method according to claim l, wherein the antibodies tosaid selected organism are bound to said magnetic solid support throughprotein A.
 3. The method according to claim 1, wherein said selectedorganism is a bacterium, yeast or mold.
 4. The method according to claim3, wherein said selected organism is a bacterium.
 5. The methodaccording to claim 3, wherein said bacterium is a member selected fromthe group consisting of Listeria, Campylobacter, Escherichia,Salmonella, Clostridia, Shigella, Staphylococci, Vibrio, Yersinia,Plesiomonas, Bacilli and Aeromonas; said yeast is a member selected fromthe group consisting of Kluyveromyces, Pichia, Saccharomyces, Candidaand Rhodotorula and said mold is a member selected from the groupconsisting of Byssochlamys, Fusarium, Geotrichum, Penicillium andScopulariopsis.
 6. The method according to claim 5, wherein saidbacterium is Listeria.
 7. The method according to claim 6, wherein saidbacterium is a pathogenic strain of Listeria.
 8. A method for detectingthe presence of bacteria which comprises:a) combining a sample to betested for the presence of a selected bacteria with a magnetic solidsupport having immobilized thereon antibodies to said selected bacteria,to thereby capture cells of said selected bacteria from said sample,said magnetic solid support being non-porous magnetic beads having adiameter of from 50 nm to 1 μm. b) exposing to a magnetic field saidsolid support having bound thereto said antibodies and capturedbacterial cells to thereby separate said magnetic solid support fromsaid sample; c) treating said magnetic beads to detect the presence ofsaid selected bacteria.
 9. The method according to claim 8, wherein saidselected bacteria is a member selected from the group consisting ofListeria, Campylobacter, Escherichia, Salmonella, Clostridia, Shigella,Staphylococci, Vibrio, Yersinia, Plesiomonas, Bacilli and Aeromonas. 10.The method according to claim 8, wherein said selected bacteria isListeria.
 11. The method according to any one of claims 8-10; whereinsaid non-porous magnetic beads have a diameter of about 1 μm.
 12. Themethod according to claim 8, wherein said antibodies are attached tosaid solid support through protein A such that the Fab portions of saidantibodies extend outward from the surface of said solid support. 13.The method according to claim 9, wherein said antibodies are attached tosaid solid support through protein A such that the Fab portions of saidantibodies extend outward from the surface of said solid support. 14.The method according to claim 10, wherein said antibodies are attachedto said solid support through protein A such that the Fab portions ofsaid antibodies extend outward from the surface of said solid support.15. The method according to claim 11, wherein said antibodies areattached to said solid support through protein A such that the Fabportions of said antibodies extend outward from the surface of saidsolid support.